Summary fluxes of free calcium ions (Ca++) are a universal regulator of biological processes and signaling in animal, plant, and microbial cells. Quantification of these Ca++ fluxes is crucial for understanding these central regulatory & signaling pathways. Currently, fluorescence-based probes are the preferred methods for measuring Ca++ levels, however these methods suffer from problems associated with fluorescence excitation, such as autofluorescence, phototoxicity, photobleaching, and poor penetration of tissue. Luminescence methods are an alternative approach that avoids these problems. This project will develop and characterize novel luminescence probes that are genetically encodable and quantify [Ca++] ratiometrically. This will be accomplished by using Bioluminescence Resonance Energy Transfer (BRET) between a luciferase and the Venus fluorophore that is modulated by Ca++-sensitive linkers so that the spectrum of emission is dependent upon Ca++ concentration. This Ca++ probe will be ideal for applications where autofluorescence, photobleaching, tissue penetration, and undesirable phototransduction & phototoxicity are problems. Using a very sensitive CCD camera coupled to a microscope through a Dual-View, Ca++-fluxes will be imaged from a variety of tissues and cells. In addition to developing and characterizing this new generation of luminescent Ca++ probes, the use of these reporters will be applied to the study of Ca++-fluxes in biological clocks (circadian rhythms). Circadian (daily) rhythms are a crucial component of human mental and physical health that regulates sleep, alertness, hormones, and many other biological processes. These rhythms are strongly implicated in some types of depression and in sleep disorders such as hypersomnia. The new Ca++ probes will be used to study circadian oscillations of Ca++ fluxes. This project is appropriate for the Exploratory/Developmental R21 Bioengineering Program because it proposes to develop new molecular probes for measurement and imaging of function as related to ubiquitous Ca++ signaling. Public Health: This project will develop new probes for Ca++ fluxes that will be optimal for conditions under which currently available methodology is limited. As a test case, these probes will be applied to the topic of biological clocks that have an important influence over mental and physical health. [unreadable] [unreadable] [unreadable]